The present application is a continuation-in-part application of my co-pending application Ser. No. 652,370, filed on Sept. 19, 1984, now U.S. Pat. No. 4,766,150 which is a divisional application of application Ser. No. 251,694, filed on Apr. 7, 1981, now U.S. Pat. No. 4,486,408.
This invention relates to a process for protecting animal cells from oxidative injury such as that caused by exposure to chemical, biochemical, or physical metabolic stressors or from inborn deficiencies in antioxidative biochemical pathways which increase cell sensitivity to oxidation induced by such chemical and/or physical factors.
This invention further relates to a process for suppressing the immune-system of animals wherein an immunosuppressive amount of 3-aminotyrosine is administered to an animal to suppress that animal's endogenous immune system.
There is a relationship between nonphosphorylating oxidative metabolism (membrane peroxidation) and immune cell activation. The production of peroxides and other active oxidants is one of the earliest measurable signals of immunogenic stimulation. Wrogemann, K., et al., 8 Eur. J. Immunol. 749-752 (1978); Strauss, R. R., et al., 15 Infect. Imm. 197-203 (1977). The antibacterial and antiparasitic activities of neutrophils (Clark, R. A. and S. J. Klebanoff, "Studies on the mechanism of antibody-dependent polymorphonuclear leukocyte-mediated cytotoxicity," 119 Journal of Immunology 1413-1418 (1977)) and eosinophils (DeChatelet, L. R., et. al., "Oxidative metabolism of the human eosinophil," 50 Blood 525-535 (1977)) are associated with peroxidative activity. Furthermore, peroxidative activity is not only associated with killing of target cells by leukocytes such as macrophages but also with cell differentiation of macrophages themselves and other cell types with which they interact.
The activators of oxidative metabolism (lipopolysaccharide and Bacille Calmette-Guerin, see Johnston, R. B. and S. Kitagawa, "Molecular basis for the enhanced respiratory burst of activated macrophages," 44 Federation Proceedings 2927-2932 (1985)) also induce tumor necrosing factor, or cachectin (see Old, L. J., "Tumor necrosing factor (TNF)," 230 Science 630-632 (1985)), and interleukin 1 (Prestidge, R. L., et. al., "Interleukin 1: Production by P388D.sub.1 cells attached to microcarrier beads," 46 Journal of Immunological Methods 197-204 (1981)). Tumor necrosing factor not only attacks tumor cells but also mediates the effects of endotoxin and the cachexia (lipolytic activity) of severe chronic infections and cancer. Interleukin 1 is necessary for committing T cells to proliferation in response to specific antigens and recruiting other lymphocytes to specific cellular or humoral responses. The latter effect is mediated by T cell production of interleukin 2.
The activation of oxidative metabolism of leukocytes is not always beneficial or innocuous. Excessive peroxidation may inhibit lymphocyte functions. Fischman, C. M., et. al., "Inhibition of lectin-induced lymphocyte activation by 2-cyclohexene-1-one: Decreased intercellular glutathione inhibits an early event in the activation sequence." 127 Journal of Immunology 2257-2262 (1981). Furthermore, excessive perixidation may activate mast cells and, subsequently, allergic reactions. Ohmori, H., et. al., "Xanthine oxidase-induced histamine release from isolated rat peritoneal mast cells: Involvement of hydrogen peroxide," 28 Biochemical Pharmacology 333-334 (1979). Autoxidation of red blood cells initiated by inflammation or chemical or physical insult may lead to autoimmune responses. Low, P. S., et. al., "The role of hemoglobin denaturation and Band 3 clustering in red blood cell aging." 227 Science 531-533 (1985).
Physical stressors that enhance peroxidation mimicking the effects of inflammation (leukocyte production of active oxygen species) include ionizing radiation and hyperthermia. It is well known that ionizing radiation of medium containing water and oxygen (i.e., tissue) produces peroxide and free radicals. Casarett, A. P. Radiation Biology, Englewood, N.J.: Prentice-Hall, Inc. (1968), Chapter 4). Phenols can act as co-oxidants forming stable phenolic free radicals. Phenol red, a commonly used pH indicator in tissue culture media, can interact with oxygen free radicals formed in vitro ionizing radiation experiments. The phenolic free radicals may, in turn, interact with other oxidizable substrates. Also, hyperthermia of red blood cells can yield superoxide and peroxide. Kiel, J. L., and D. N. Erwin, "Thermochemiluminescent assay of porcine, rat, and human erythrocytes for antioxidative deficiencies," 143 Analytical Biochemistry 231-236 (1984); Kiel, J. L. and D. N. Erwin, "Microwave and thermal interactions with oxidative hemolysis," 16 Physiological Chemistry and Physics and NMR 317-323 (1984). The peroxidative reactions associated with inflammation, ionizing radiation, and hyperthermia may result in membrane protein crosslinking (Karel, M., "Lipid oxidation, secondary reactions, and water activity of foods." In M. G. Simic and M. Karel (eds.), Autoxidation in Food and Biological Systems, pp. 191-206, New York: Plenum Press (1980)), cell lysis (Lynch, R. E., and I. Fridovich, "Effects of superoxide on the erythrocyte membrane," 253 Journal of Biological Chemistry 1838-1845 (1978)), oxidation of proteins or enzymes with loss of function (Karel, M., supra), nicking DNA (Lown, J. W., S. K. and Sim, "The mechanism of the bleomycin induced cleavage of DNA," 77 Biochemical and Biophysical Research Communications 1150-1157 (1977)), and inhibition or activation of inflammation by the specific production of prostaglandins or leukotrienes (Goldstein, I. M., et. al., "Thromboxane generation by human peripheral blood polymorphonuclear leukocytes," 148 Journal of Experimental Medicine 787-792 (1978); Weissman, G., et. al., "Prostaglandins and inflammation: receptor/cyclase coupling as a explanation of why PGE's and PGI.sub.2 inhibit functions of inflammatory cells," 8 Advances in Prostaglandin and Thromboxane Research 1637-1654 (1980); Samuelsson, B., et al., "Introduction of a nomenclature: Leukotrienes," 17 Prostaglandins 785-787 (1979); and Zurier, R. B. and G. Weissman, "Effect of prostaglandins upon enzyme release from lysosomes and experimental arthritis," In P. W. Ramwell and B. B. Phariss (eds.) Prostaglandins in Cellular Biology, pp. 151-172, New York: Plenum Press (1972)).
Therefore, any inhibitor of early peroxidative events would serve as a profound immunomodulator and anti-inflammatory agent. Dose-dependent effects of such inhibitors are expected; that is, protection without inhibition of the immune response at low doses and immunosuppression at high doses. Also, other tissues affected by autoxidation and peroxidation (i.e., red blood cells) would be protected by such an inhibitor.
As noted above, the autoxidation of red blood cells (RBCs), which may be initiated by such stimuli as inflammation or chemical or physical insult, may lead to autoimmune responses. However, the autoxidation of RBCs is a common occurrence even under normal conditions. In other words, such stimuli are not required to initiate autoxidation. As much as 3% of the total hemoglobin (the hemoglobin being contained within the RBCs) is converted to methemoglobin each day, an event which results in the formation of superoxide, hydrogen peroxide, and lipid peroxides, all of which are oxidants which pose a significant threat to all cells, but especially the RBCs, where the event occurs.
Under normal circumstances, the oxidants generated during autoxidation cause little damage to the RBCs due to the scavenging action of several enzymes. These enzymes include superoxide dismutase, which catalyzes the conversion of superoxide to hydrogen peroxide and molecular oxygen, and catalase and peroxidase, which catalyze the conversion of hydrogen peroxide to water in the presence of available electrons. The peroxidase involved in autoxidative pathways, at least in known mammalian cells, is glutathione peroxidase, which is a selenium-containing enzyme that oxidizes reduced glutathione and reduces peroxides.
Inherited deficiencies of superoxide dismutase and catalase, or deficiencies in key co-enzymes, may lead to increased susceptibility to oxyhemoglobin autoxidation, as may the inhibition of those enzymes. Shanus, supra; Metzler, D. E., Biochemistry: The Chemical Reactions of Living Cells, New York: Academic Press (1977), pp. 564-565. Disorders such as malignant hyperthermia have been associated with inherent glutathione peroxidase deficiency in swine. Schanus, E. G., et al., "Malignant hyperthermia (MH): Porcine erythrocyte damage from oxidation and glutathione peroxidase deficiency," in G. J. Brewer (ed.), The Red Cell, Fifth Ann Arbor Conference, New York: Alan R. Liss, Inc. (1981), pp. 323-336. The addition of various anionic nucleophiles (i.e., azides, halides and thiocyanate) to the cells accelerates the autoxidation reaction. Wallace W. J., et al., "The mechanisms of hemoglobin autoxidation: Evidence for proton-assisted nucleophilic displacement of superoxide of anions", 57 Biochem. Biophys. Res. Comm. 1104-1109 (1974). Increasing the temperature to which the cells are exposed by 3.degree. C. doubles the rate of oxyhemoglobin autoxidation under physiological conditions. Wallace, W. J. et al., "A role for chloride in the autoxidation of hemoglobin under conditions similar to those in erythrocytes." 43 FEBS Letters 33-36 (1974).
Further, ample evidence has shown that the combination of certain peroxidases with hydrogen peroxide and a halide ion produces a system with strong cytotoxic properties. The myeloperoxidase-hydrogen peroxide-chloride system forms a potent cytotoxic system effective against bacteria, fungi, viruses, mycoplasma, and various mammalian cells. Similarly, the lactoperoxidase-hydrogen peroxide-thiocyanate system and the horseradish peroxidase-hydrogen peroxide-chloride system have been shown to have potent cytotoxic activities.
An equally cytotoxic system is obtained when instead of hydrogen peroxide, a hydrogen peroxide generating system is used. Thus, the glucose oxidase-horseradish peroxidasechloride combination yields a potent cytotoxic system upon the addition of glucose. Galactose oxidase and xanthine oxidase have also been shown to be effective in this respect. Furthermore, we showed that the endogenous NADH oxidase activity of horseradish peroxidase is also capable of promoting the cytotoxic activity of the enzyme in the presence of chloride ions.
A large body of evidence indicates that cytotoxic systems such as those described above may be operative in polymorphonuclear leukocytes, eosinophils, macrophages, and other cell types with cytotoxic properties. Such cells in general appear to utilize an NADH or NADPH oxidase as the peroxide-generating enzyme.
Macrophages are a necessary component in the augmentation of natural killer cell activity by Bacillus Calmette-Guerin (BCG) in mice. BCG also increases the peroxide and superoxide production by macrophages. The possibility thus exists that the peroxidase system of the macrophages plays a role in the augmentation of the natural killer cell activity. Similarly, peripheral lymphocytes, which are predominantly T-cells, contain a cytotoxic peroxidase. Chemiluminescence resulting from peroxide generating oxidative metabolism is observed when T lymphocytes are stimulated by concanavalin A. Furthermore, immunization of mice with either soluble or particulate antigens causes an increase in peroxidase activity in the spleen which precedes the generation of specific antibody. These observations suggest that oxidase and/or peroxidase activity is in some way involved in developing specific immune responses.
Thus far none of the cytotoxic systems described above have been used in any in vivo experiments. However, some relevant experiments were done some time ago by Schultz and his colleagues. Schultz, Snyder, Wer, Berger and Bonner, "Chemical Nature and Biological Activity of Myeloperoxidase," in Molecular Basis of Electron Transport, New York: Academic Press, (1972), pp. 301-321 (1972); Schultz, Baker, and Tucker, "Myeloperoxidase-Enzyme-Therapy of Rat Mammary Tumors," in Cancer Enzymology, New York: Academic Press (1976), pp. 319-334 (1976). Using mice bearing 20-methylcholanthrene induced tumors, these authors injected myeloperoxidase in combination with thio-TEPA, an antitumor drug. They observed a significant reduction in tumor growth in the treated mice, but no complete remissions. Neither myeloperoxidase nor thio-TEPA alone were effective in reducing tumor growth. The inhibition of tumor growth lasted as long as the treatment with myeloperoxidase and thio-TEPA was continued.
These results indicated that the activity of myeloperoxidase could play a role in the control of tumor growth, either directly or indirectly. Definite conclusions are difficult to obtain with such experiments, however, because the biological half-life of myeloperoxidase is only about 24 hours. It is noteworthy that the toxic activity appeared to be specifically directed to the tumor tissue.